The goal of this project is to define the interactions between CD34-expressing hematopoietic progenitor cells (HPCs) and human cytomegalovirus (HCMV) that impact viral latency and reactivation. HPCs are a prominent site of HCMV latency and a common target of human viruses. Latency enables HCMV to persist indefinitely in infected individuals and prevents pathogen clearance. Reactivation of HCMV from latency in immunosuppressed individuals, including stem cell and solid organ transplant recipients and AIDS patients, is associated with high rates of morbidity and mortality. Despite its critical importance to HCMV pathobiology, the mechanisms underlying HCMV latency remain obscure. Elucidating the mechanisms of viral latency in HPCs will contribute importantly to our understanding of viral disease in immunocompromised individuals. Latency- competent clinical strains of HCMV contain a unique 13-15 kilobase (kb) region of the genome, termed ULb', that has been lost from strains adapted for replication in cultured fibroblasts. These laboratory-adapted strains cannot support a latent infection. We hypothesize that HCMV encodes viral determinants of latency within the ULb' region of the genome that function in latency through interactions with other viral or cellular proteins. Using a novel experimental model we developed to recapitulate latency in primary human HPCs infected in vitro, we have identified a 5 kb region, and more specifically the UL138 open reading frame (ORF), within the ULb' region of the genome that is required for a latent HCMV infection in HPCs. The UL138 ORF has not been previously characterized with respect to HCMV infection or latency and represents the first functional viral determinant of latency. We propose to determine the function of UL138 in promoting a latent infection. In Aim 1, we will identify and characterize the gene products derived from the UL138 locus that are required for a latent infection. Our preliminary studies have identified two large transcripts and a protein encoded by the UL138 gene locus. In Aim 2, we will identify novel viral determinants of latency encoded in the ULb' region and determine which determinants are sufficient to restore latency to a latency-incompetent recombinant strain of HCMV. In Aim 3, we will elucidate the mechanism by which the UL138 protein and other determinants promote latency by identifying viral and cellular proteins that interact with UL138. HPC-specific interactions will be functionally characterized by analyzing the impact of their expression on HCMV latency and replication. Viral and cellular proteins that contribute to HCMV latency will implicate cellular pathways exploited or negotiated by the virus in HPCs. These studies will define key mechanisms governing HCMV latency. Further, this work will identify candidate molecular targets leading to improved antiviral therapies aimed against latently infected cells to prevent HCMV disease and complications associated with reactivation of latent HCMV.